CA2148479A1 - Fibronectin binding protein as well as its preparation - Google Patents

Abstract

2148479 9410330 PCTABScor01 The present invention relates to a new fibronectin binding protein from E. coli in the form of a curli pili, a new recombinant hybrid-DNA-molecule comprising a nucleotide sequence from E.
coli) coding for a protein or polypeptide having fibronectin binding properties.

Description

WO94/10330 21~8l7 l PCI/US93/~0547 FIBRONECTIN BINDING PROTE~N AS WELL AS lTS PREPARATION

DES~RIPTION
Technical Fiekl ~ ~-The present invention relates to a fibronectin binding pro~in as well as S a hybrid-DNA-molecules, e.g., plasmids and phages comprising a nucleotide sequence coding for s~id protein. Further the invention relates to microorganisms comprising said molecules and t'neir use producing said protein, as well as the synthetic preparation of s~id protein. ~ -The object of the present invention is to obtain a minimal fibronectin -~
binding protein. -A further object of the present invention is to obtain said protein by means of a genetic engineering technique by using, e.g., a plasmid comprising a nucleotide sequence coding for said pro~ein. ~ -A further object is to obtain a possibility of preparing said protein by chemical synthesis.
Further objects will be apparent from the following description.

Back~round of the Invention WO-Al-85/05553 discloses bacteAal cell surface proteins having fibronectin, fibAnogen, collagen and/or larninin binding ability. Thereby it is shown that different bacteria ha~e an ability to bind to fibronectin, fibrinogen, `
collagen and/or laminin. It is further shown that fibronectin binding protein ~
derived from StaphYlococcus aureus has a molecular weight of 165 1~1) and/or ~-87 kD, whereby it is probable that the smaller protein is a part of the larger one.
Fibronectins are a family of high molecular weight glycoproteins occumng in a soluble fonn in many body fluids and in an insoluble form as a compound of cell surfaces, basement membranes, and extr~cellular ma~ices. 5 Fibronectins appear to fulfill a critical role in clearance by phagocytes of autologous tissue debris, immune complexes, and bacte~ia. ~iibronectins also ~3UBSTITUTE S~EEl Wo 94~ l 0330 Pcr/uss3/1 0~47 21~ 1 r~

.
bind to epithelial cells. In doing so it may serve as a receptor for organisms ~ `
like group A streptococci, but may also shield the epithelial receptors for , ,~
other organisms. Thus the inability of Gramnegative organisms like Ps. ~ ~`
aeuruginosa to colonize tbe oral cavity of healthy humans may be due to an S interference in binding to epithelial receptors by fibronectin. ~The ability to ~ -resist binding to soluble fibronectin has been thought to be a virulence factor i~ `~
in invasive infection by group B streptococci. A number of Grampositive bacterial species including Staphvlococcus aureus, other staphylococcus -species, and group A, C and G streptococci exhibit specific interaction with ~--fibronectin. In these species binding to fibronectin is thought to be a virulence factor enhancing colonization of wound surfaces and other fi~
bronectin coated surfaces. E. coli can express a variety of adhesins with `
diffeAng binding specificities. The majority of these adhesins recogni~
~ .
carbohydrate moieties present on glycoconjugates. E. coli may also express -binding to matrix proteins such as fibronectin, laminin, and collagen.
Uropatogenic E coli expressing the 075 X adhesin bind tubular basement membranes and to the Bowman capsule known to be rich in laminin. The purified 075 X adhesin was specifically found to bind laminin. E. coli isolated from patients with ulcerous colitis frequently bind matri~ proteins.
Likewise, E. coli isolates from bovine mastitis ha~e been shown to bind to fibronectin at a high frequency. Below a native fibronectin binding protein from E ~QIi is disclosed, as well as the cloning of the fibronectin binding, I ` -f~ gene from a bovine isolate of E. coli that express eurli pili and fibronectin binding when present in E. ~ HB101. ; ~ `-`
2S Chemically fibronectin is a large glycoprotein (Mt about 450 kD) with~wo similar subunits, which may vary in molecular si~e depending on a complex splicing pattern of a precursor MRNA. I~e major function of fibronectin which is found in body fluids blood ciots and e~tracellular matrices ¦
, ~
scems to be related to the ability of the protein to mediate substraee adhesion i- ~ -~
of most eukaryotic cells. i - -." ,....

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Descr;ption of the Present Invention It has now surprisingly been found possible to obtain a native fibronectin binding protein from E. coli, as well as a hybrid-DNA-molecule comprising a nucleotide sequence coding for a protein or a polypeptide having fibronectin binding properties. As evident from below the following -nucleotide sequence is present in the gene coding for said protein:

AGGTGTTGTT CCTCAGTACG GCGGCGGCGG TAACCACGGT GGTGGCGGTA :~
ATAATAGCGG CCCAAATTCT GAGCTGAACA TTTACCAGTA CGGTGGCGGT -~
AACTCTGCAC TTGCTCTGCA AACTGATGCC CGTAACTCTG ACTTGACTAT ~:~
T~CCCAGCAT GGCGGCGGTA ATGGTGCAGA TGTTGGTCAG GGCTCAGATG
ACAGCTCAAT CGATCTGACC CAACGTGGCT TCGGTAACAG CGCTACTCTT
GATCAGTGGA ACGGCAAAAA T~CTGAAATG ACGGTTAAAC AGTTCGGTGG
TGGCAACGGT GCTGCAGTTG ACCAGACTGC ATCTAACTCT CGTCAACGTG
ACTCAGGTTG GCTTTGGTA :~

corresponding to the following ammo acid as identified from the N-terminal .
end G W POYGGGG NHGGGGNNSG PNSELNTYOY GGGNSALALQ TDARNSDLTI
TQHGGGNGAD VGQGSDDSSI DLTQRGFGNS ATLDQWNGKN SEMTVKQFGG
GNGAAVDQTA SNSRQRDSGWLW* ' : .
- ' 20 wherein A Alanine R Arginine N Asparagine D Aspartic acid C Cysteine Cystine G Glycine E C~lutamic acid Q Glutamine H ~Iistidine ' ::

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Isoleucine T Leucine ', K Lysine M Methionine F Phenylalanine P Proline S Serine T Threonine W Tryptophan Y Tyrosine V Valine * stop codon `
The mature curlin protein has a molecular weight of 17 ~D when gel- ~;
purified, and when ~etermined via the nucle~tide sequence it is predicted to contain 122 amino acids providing ~or a molecular weight of 14,345 dalton.
The invention further comprises a plasmid or phage comprising a nucleotide se~uence coding for said fibronectin binding protein, T'he invention further compnses a micro-organism comprising at least one hybrid-DNA-molecule according to the above. The microorganism, E.
coli HB101/pPnb~0 encompassing the plasmid encodin~7 for said nucleotide sequence was deposited May 05, 1988 at Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, FRG under the deposition number DSM 4585.
The invention further comprises a method for producing a fibroneetin binding protein whereby at least one hybrid-DNA-molecule of above is r introduced into a micro-organism, cultivating said micro-organisrn in a growth medium, and isolating the prot~in thus formed and expressed by means of an affinity chromatography on a fi~ronectin bound to an insolubilized camer followed by ion exchange chromatography.

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A further aspect of the invention comprises a chemical synthesis of the fibronectin binding protein, whereby an amino acid sequence is built up based on said nucleotide sequence encoding for said protein starting from the C- ;
terminal amino acid which is stepwise reacted with the appropriate amino S acid, whereby it is finally reacted with the amino acid at the N-terminal end, to form ~he fibronectin peptide region.
~ppropriate carrier proteins can be coupled to the amino acid sequence ~as well, such as IgG binding regions of protein A.
The invention will be descAbed in the following with reference to the example given below? however, without being restncted thereto.

Example Isolation and characterization of a recombinant clone expressin~ fibronectin In a collection of bovine faecal and mastitis isolates of Escherichia CQli, 58% and 56%, respectively, bound to ~25I-labelled ~lbronectin. In some of the mastitis isolates the ability to bind to ~1bronectin was not a stable property. Thus a chrornosomal cosmid bank was generated from faecal E
~1~ isolate AO12, sinc~ that strain stably e~pressed binding to fibronectin.
Such binding was preferentially expressed when the cells were grown on CPA-agar at temperatures between 26C and 32C. Electron microscopy of 40 fibronectin binding E. coli. isolates revealed that they all produced coiled surf~ce structures when grown on CFA~agar at 26C. High magnification of these structures showed them to be thin, wiry fibers with a diameter of about 2 nm. The lateral aggregahon of individual fibers produced considerably thicker structures. ~;
Bacteriophage transducing particles carrying portions of the strain AO12 genome cloned into cosmid vector pJB8 were used to transfonn E. coli HB101. ~train-AO12 expressed fibronectin binding when grown on CFA
plates at 30C, but to a lesser extent at 37C. Transductants were thus :.

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screened for fibronectin binding after growth on CFA plates for 40 hrs at 30C. Out of 560 transductants one, A0450, showed fibronectin binding.
The recombinant cosmid in this transductant, designated pA0450, was isola~ed , -and shown to contain an insert of about 24 kb. pA0450 was digested with 5 restr~ction endonuc}ease SalI and subcloned into the Vector pACYCl84, giving pPnbOl. A subclone of pFnbOls viz. pFnblO containing a 3 kb large , SphI-KpnI fragment expressed fibronectin binding when transformed into E.
coli HB101. The subclone pFnblO was digested with a series of restnction endonucleases to prepare a restriction map as shown in ~G. 1. To further 10 localize the region on pFnblO expressing fibronectin binding several subclones were constructed from pFnblO as were various deletion derivahves. These cons~ructs were tested for their ability to confer binding to E. coli HB101.
The HpaI and SphI sites delineates the 1.2 kb region required far fibronectin `~
binding as shown in FIG. 1.
The recombinant plasmids shown in FIG. 1 were transformed into the minicell producing strain AA10. Plasmid encoded polypeptides were analyzed from 35S-methionine labelled minicells. Plasmid pFnblO expressed hvo polypeptides with molecular masses of 43 l~D and 17 kD respectively as shown in FIC; 2. pFnb30 is a deletion derivative of pFnblO that lacks a 0.9 20 kb ClaI fragment. This derivative no longer confer fibronectin binding to E.
~Qli IIB101. The 49 kD polypeptide was still expressed from this clone but the smaller 17 kD polypeptide was missing. The larger polypeptide ~ust be .
encoded from a gene positioned between the C~lal2 and KpnI sites on pFnblO, since the region between SphII and ClaIl site is to small (0.4 kb) to encompass .`
25 a gene coding for a 49 kl~ protein. It is aIso concluded that the 49 kD
polypeptide is not required for fibronectin binding. Plasmids pFnb20 and ~;
pPnb58 both expressed fibronectin binding in E. ~i. The 17 l:D polypeptide but not the 49 kl~ polypeptide was encoded by pFnb20 showing that the gene ~ /, for the Iatter polypeptide must span the SphI2 site. The HpaI-SphI2 subclone 30 pPnb56 no longer expressed t~e 17 kD polypeptide.

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Instead a slightly larger polypeptide species of 19 kD was expressed. I -An 1.5 kb large fragment carrying the aminoglycoside 3 phosphatransferase gene plasmid of pUC-4K was cloned into the single ClaI site of pFnb56 giving ! :
pPnbS9~ This derivative no longer mediated fibronectin ~inding. In minicells 5 there was no expression of the 19 kD polypeptide. The conclusion is that the 19 kD polypeptide is due the deletion in the pYnbS6 of the normal translational stop codon of the gene for the 17 ~D polypeptide giving ~ise to a slightly longer translational product containing some carboxyterminal amino acids encoded by the vector.
The coiled surface structure could be shared from the surface of HB101/pFnb20 in an omnimixer and such partially purified preparations contained a dominating protein species with a molecular weight of 17 kD.
The 17 kD polypeptide was elektroeluted onto an ImmonobulonR filter and the aminoterminal sequence determined by sequential Edman degradation. In 15 order to deterrnine if the isolated protein was the same 17 k D gene product of the structural gene spanning~the ClaI, and the H~3I sites in pFnb20 this region was sequenced. One open reading frame consisting of 133 codons was identified spanning the ClaI and ~3I sites. The protein se~uence of the S
amino tenninal residues was identical to the DNA sequence specified by 23 codons 2-6 in the open reading frame confinning that the open reading frame identified encodes the subunit protein of the coiled surface structure. The `
name "curlin" is proposed for this subunit protein, curli for the structure and csga for the structural gene. ~ -~
The curlin subunit appears to be strictly di~ferent from E. coli pilins 25 and E. coli pilins have several features in common such as cleavable signal peptide, two cysteine in the amino-terminal half, and several conserved amino acids in the amino and carboxy terminal regions thought to be involved in the subunit interactions. Likewise no homologoes were found with the N-methyl ~ !
Phe class of piIus expressed by Neisseria ~onorrhoeae and many other Gram 30 negative species. Flagellin is not a true secretary protein since it is : .

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transported directly from the cytoplasm to ~he growing tip through the hollow flagellum. The small diameter of the curli suggest that they are assembled from the base. Hence expression of pili is not possible in E. coli HB101 when only the major pilus subunit protein is introduced into thsi-strain. In 5 contrast curlin is able to polymerize into curli in the absence of any other cloned gene products.
Furthermore the data implicates that the gene for the 17 RD po- ~-lypeptide spans the ClaI2 site on pFnblO. Since fibronectin bmding was mapped to this gene it was denoted fnbA. E. coli pilin proteins are all known 10 to be produced in a precursor form with a cleavable N-tenr~inal signalpeptide.
To see if the Fnb protein was synthesized from ~nbA in a precursor form, minicells expressing Fnb from plasmids were treated with 9% ethanol to prevent processing. There was no appearance of a large molecular weight species suggesting that Fnb is not synthesized with a cleavable signalpeptide.

15 Nucleotide sequence of fnbA ~ene Sequence analysis of a CsCl2-purified double-stranded plasmid DNA
from pCSG4 was performed by denaturing approximately 4 ug of DNA with 2 M NaOH/2 mM EDT~ and neutralizing it with 7.5 M ammonium acetate (pH
5). Appropriate oligonucleotides (1 pmole) were armealed to aLkalidenatured ~0 DNA and sequenced using the Sequenas~ protocol as desc~ibed by the manufacturer (United States Biochemical, Clevdand, OH). Electrophoresis was pefforrned in a 90 mM ll~E buffer system for 2-5 hra at 45 mA in 8 M
urea/6% polyacrylamide gels. Gels were fixed, dried, and exposed to Hyperfilm ~Amersham).
i~, i,, .

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., WO 94/lO~S30 PCl/US93/105~7 ~`~ 2 ~ d ~ 3 BGGTGTTGTT CCTCAGTACG GCGGCGGCGG TAACCACGGT GGTGGCGGTA
ATAATAGCGG CCCAAATTCT GAGCTGAACA TTTACCAGTA CGGTGGCGGT
AACTCTGCAC TTGCTCTGCA AACTGATGCC CGTAACTCTG ACTTGACTAT
~ACCCAGCAT GGCGGOGGTA ATGGTGCAGA TGTTGGTCAG GGCTCAGATG
S ACAGCTCAAT CGATCTGACC CAACGTGGCT TCGGTAACAG CGCTACTCTT
GATCAGTGGA ACGGCAAAAA TTOTGAAATG ACGGTTAAAC AGTTCGGTGG
TGGCAACGGT GCTGCAGTTG ACCAGACTGC ATCTAACTCT CGTCAACGTG
ACTCAGGTTG GCTTTGGTA

This will correspond to the following amino acid sequence GVVE'OYGGGG NHGGGGMNSG PNSELNTYOY GGGNSALALQ TDARNSDLTI
TQHGGGNGAD VGQGSDDSSI DLTQRGFGNS ALTDQWNGKN SEMTVRQFGG
GNG~AVDQTA SNSRQRDSGWLW

Expression of a curli pili from the original strain A012 and from HB101 clones expressing the Fnb protein. .
lS ~ coli strain AO12 grown at 26C expressed ~musually curli pili-like structures when viewed under the electron microscope as shown in FIG. 4.
No such structures can be seen on E. coli HB101 growing at either 26C or ...
37C. However, cells of HB101 harboring either pFnb20 of pFnb56 produced large amounts of curli pili structures when grown on CFA medium 20 at 26C as shown in FIG. 5. However, no surfacc structures was found on ~IB101/pFnb20 and HB101/pFnbS6 after growth at 37C as shown in FIG. 6, a temperature at which these strains do not express fibronectin binding. No surface structures were found on HB101/pFnb56 deleted for the ~_a gene ~.
when grown at either 26C or 37C.
Hence the expresslon of curli pi1i in HB101 was strictly correlated to ~he expression of the Fnb protem. Moreover, the expression of curli pili correlated strictly with the ability of HB101 to bind to fibronec~in. ~~
~ coli HB101 has been used as a host to clone and express a number 7 ``
of E. ~ pili types. In no other case it has heen possible to obtain surface ~ i~
located pili by only expressing the pilin gene. All other hlown gene cluster !:

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that have been exarnined today contain additional genes required for ~ansport and assembly of the pilus fiber. When plasmid pFnb20 was present in ~ coli AA10 curli pili were not observed at 26C as shown in FIG. 7 and the cells did not bind fibronèctin. Most F ~Li strains carry in their chromosome, genes for type 1 pili. Both E. coli HblO1 and AA10 are unable to form such pili. In the former strain some genes from the type 1 operon are still functioning whereas the entire type 1 gene cluster is deleted in AA10. -Plasmid pSJ~9 carries all accessory genes required ~or type 1 pilus formation but is deleted for the fimA gene encoding the major pilin subunit. E. ~
AA10 harboring plasmid pSJH9 and pFnb20 did not bind fibronectin. Hence the ability of pFnb20 to express fibronectin binding and curli pili in HB101 does not seem to depend on the comnlementation from chromosornal type 1 pilus genes. To see if accessory genes located close to fnba could confer fibronectin binding to E. coli AA10 this strain was transformed with the original cosmid clone pA0450~ Strain AA10/pA0450 was not expressing fibronectin binding. Minicell experiments revealed that s~ain AA10 is able to synthesize the FnbA protein at 26C as well as at 37C. It IS therefore concluded that the FnbA protein cannot be expressed for surface loealization, nor be assembl~ into curli pili in this strain.
Escherichia Ç~li is a common cause of bovine mastitis. L coli n~ilk 1, isolates from cases of acute mastitis do not differ from the normal faecal E.
~Qli flora in the cow. When examining possible virulence asso~iated properties of bovine E. coli the ability to bind to fibronectin stands out as a common property shared by 50-80% of the isolates studied To more closely study fibronectin binding in E. coli the fnbA gene was cloned and characterized. The fnb gene codes for a 17t~ dalton protein that i5 able to polymerize into curli pili-like structures when expressed in E. coli HblO1.
It is surpnsing that DNA fragment expressing the FnbA protein, only, could confer piliation in HB101. E. coli pili are encoded from an operon consisting of 11 ~2 genes. The accessory genes ~C coding for an 88 kD

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~ ., outer membrane pore protein, and ~D expressing a 28 kD periplasmic transport protein are needed in addition to ~A, the major subunit gene to form surface located pili. Even though other classes of pill adhesin i genecluster appear to be less complex and.contain fewer genes than the system all carry genes functionally equivalent to ~C and E~D. -The fact that the fnb plasmid p~nb20 did not confer piliation or curli ;
pili when harbored by E. ~. AAlO suggests that E. ~li HBlOl express the accessory proteins required for the biogenesis of curli Fnb pili. It is known that E, coli HBlOl ca~Ty some DNA that hybridize to cloned type 1 DNA.
However, E, ~ HBlOl do not express type 1 pili if transformed with a type ~ ~ -1 clone deleted for either the fimD (the ~C equivalent) or the fimC (the ~D equivalent) gene. It is therefore unlikely that it is chromosomal type 1 ' ` -DNA that encodes the accessory proteins required for the formation of curli Fnb pi}i. This is further supported by the observation that AAlO cells expressing both the Fnb protein and the accessory of the type l did not -express fibronectin binding Fnb pili. Strain AAlO is a minicell producing strain. The Fnb protein was clearly synthesized in the minicells although no pili were formed. In the ~ system a similar phenotype would have been obtained by mutating the ~C gene. Mutations in the ~2D gene results in a rapid proteolysis of PapA pilin. If similar accessory genes were at hand in ~ -the fn_ system it could be argued that E. coli AAlO lacks a ~C equivalent ~ -so that the Fnb protein cannot be surface localized.
Curli Fnb pili are formed in HBlOl at 26C but not at 37C. In AA10 minicells the Fnb protein is synthesized to the same extent a~ both ~ `
2~ temperatllres. It is thus suggested that the temperature regulation of piliation and fibronectin binding do not operate at the level of transcription but at the ~s level of pilus biogenesis. Pap pili and many other virulence associate~
properties are also temperature regulated but the effect of incubation tempera~
ture is the reYerse of what has been observed concerning the Fnb pili. In 30 these former system transcription is increased with an increased temperature~
~3UE~STITUTE SHEET

' .'' WO 94/10330 PCI~US93/10547 21~8 l~.`3 This kind of temperature regulation is thought to reflect the adaptation of the ~' ;
rnicro-organism to the mammalian host. i Wound pathogens such as Staphylococcus aureus and Staphylococcus genera frequently bind to fibronectin. The temperature of the skin is lower than 37C. If fibronectin binding of E. coli also reflects an adaptation to bindto wounds on the exterior of the host it would be an advantage for the micro-organism to have optimal binding at a temperature lower than 37C.
Hereirl there is no evidence given for the fact that fibronectin binding ~:
curli pili actually plays a role in the pathogenesis of bovine mastitis. Ihe frequent presence of ulcers, due for example to rnachine milking on the udder might, however, enable colonization of fibronectin binding E coli. ~`
Fibronectin is a large glycoprotein lalown to bind to a large set of proteins. It is therefore uncertain how specific the interaction is between fibronectin and Fnb pili. The fnbA cosmid pAO450 was the single one out of 560 cosmids tested that expressed fibronectin binding in HB101. It is thus believed that this gene is the sole determinant of fibronectin binding in E. coli AO12.
, Materials and methods.
Bacterial strains and ~rowth conditions.
Except for the bovine isolate AO12 aIl bacterial strains are deriva~ives of E. coli K~12, HB101, sdr, hsdm-, recA13, supE44, lacZ4, leuB6, proA2, thr-1, Smr) were used as host in the fibronectin binding assay. JM83 (~, aa~-~AB), E;e L, 80, làcZ M15) were used as host in all 3 transformation experiments. For prote~n expression analyses a recA denvative . ~ `
of P678-54 (Lund et al, J. Baçt. ~, 1293-1301), AA10 ~obtained from `
P. Orndorf, Stanford University, Stanford, California) was used. M~3 ~ :
cloning and phage propagation were carried out in strain ~M103 (Messing et al., ~ 9:309-321). Strain A012 is a bovine faecal isolate obtained ~rom a healthy cow.

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Other E coll K12 strains were tested and analy~ed for the production I ~
of curli and the ability to bind fibronectin. Strain C600 ~-1 leuB6 lacYl ! ~`
suoE~4 rfbDl thi-l tonA21) was found to bind fibronectin when grown on CFA-agar at 26C, at which temperature it also produced curli. The ~EQA2 S deletion mutant A31157 did not, as expected express neither property. The mir~lcell producing E. çQ~ K12 strain AA10 carries the crl region as revealed ~ -~
by DNA-DNA hybridization using the 1.5 kb large BglII-$phI fragment as a probe, but did not express curli and did not bind soluble ~lbronect~
Bactena were normally grown in L-broth ~Bertani l9Sl). For the 10 fibronectin binding assay bacteria were grown on CFA-agar (Evans, InfL and Imm., 25, 738-748) and containing 0.005% magnesium sulphate and 0-0005%
magnesium chloride in 2% Bacto agar (I)ifco). Competent cells for transforrnation were prepared with 50 mM CaCI~ 6, 23-28, Dagert).
The antibiotics ampicillin (100 / ug/ml), kanamycin (50 / ug/ml) and 15 chloramphenicol (20 / ug/ml) were used for selection of plasmid-containing s~ains. Unless otherwise stated, incubation of bacterial cultures was carried j i out at 37C. -The present fibronectin binding protein will sometimes be synthesi~d in the cell walI and is not expressed as a pili. This protein thus synthesized can be isolated as well by known biochemical methods, such as affinity chromatography .

Rec~mbinant DNA ~echni~ue. .
Reistriction endonucleases, T4 ligase and Sequence~' were used ~ ~
according to the condi~ons recommende~ by the manufacturers (New ? `;
England, Biolabs, PL, Ph~acia, Uppsala7 Sweden; US13, Cle~eland, Ohio, s~
USA).
Isolation of plasmid DNA, agarose gèl elec~ophoresis, transforma~on of E. ~Q!i and isolation of DNA fragments from polyacrylamide gels were pe~fonned essentially as described by Maniatis et al ~198~). Relevant ' `
' ~:

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2 1 A 8 ~ 7 ~, i - 1 4- j fragments were subcloned into M13mplB and Ml3mpl9 vector (Messing, J ~ -and Vieira, J. Gene., 19, 269-272 (1982)), and sequenced using the dideoxy chain terminating method of Sanger et al, PNAS, 7¢, 5463-5467. For DNA
sequencing the bacteriophage 1~7 DNA polymerase, and Se~uenaseTM were S used. The primer used was Universal M13 17rner and synthesized 20mer oligonucleotides supplied by Symbicom, Umea, Sweden. ` `

..~.',-Isolation of chromosomal and plasmid DNA.
~A was isolated as described by Lund et al. Plasmid DNA from clones carrying recombinant DNA was isolated by the alkaline lysis procedure '`
(Maniatis, CSH, N.Y., USA).

Cosmid cloni~ procedure.
Chromosomal DNA purified frorn E. coli A012 were partially cleaved with endonuclease Sau3AI. The DNA was size fractionated on a 10-40%
linear sucrose gradient. Fractions containing DNA fragments larger than 20 kb in size were pooled and ligated into the BamHI site of the cosmid vector pJB8 as described by Maniatis, CSH Lab, N.Y., USA. Recombinant -molecules were packaged in vitro into particles using a lambda DNA in vitro l~;
packaging kit (code N.334, Amersham). I~e phage was then used to infect E. coli HB101 by selecting for ampicillin resistant clones after growth on ~-CFA agar plates at 30C for 40-48 hrs.
, Plasmid construction ~ -The cosmid clone pA0450 carried a roughly 24 kb large chromosomal insert. Plasmid pFnbOl was constructed by subcloning a 4.9 kb large SalI
Ç:; -.-. .
fragment from this cosmid into pACYC184. An intemal 3 kb large SphI~-25 KpnI from pPnbOl was cloned into pUC18 (Messing) giving pFnblO. A
number of subclones from pFnblO were generated by cloning into the polylinker site in pUC18. Plasrnid pFnbS9 is a ClaI cutback deAvative of ~3iU~3STlTVTE i~1EE~T ':

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pPnblO. Subclones were constructed as follows. pFnblO consists of the 3.0 ' `
kb KpnI-PstI fragment of the original clone Iigated into the polylinker cloning cassette of pUC18, while pFnb30 is a ClaI deletion derivative of pPnblO.
Plasmid pFnb20 was obtained by cloning the 1.5 kb SphI-BglII fragment from S the original plasmid into pUClg. Analysis of this construct which lacks the 5'terminal end of the gene coding for the 49 kD peptide showed that this protein was not necessary for curli production or for fibronectin binding. ~Plasmids pFnbS6 and pFnb46 are SphIHpaI and ClaI~lII subclones, respectively of pFnblO in pUC18. Plasmid pFnb59 was obtained by first subcloning the KpnI-B~lII fragment of the original plasmid in plJC18 and then inserting the ;
aminoglycoside-3'-phosphotransférase gene (APH) from the mobilization plasmid pUC4K into the ClaIl site. To perform the fibronecdn binding assay bacteria were grown an CFA-agar plates for 42-48 hrs at 26C or 37C.
AccI digested plasmid pUC-4K (Pharmacia, Uppsala, Sweden) carrying the kanamycin resistance gene from transposon Tn903 coding for aminoglycoside 3 phosphotransferase (APH) was ligated to ClaI digested pFnb56. Transforrnants in HB101 were screened for ampicillin and kanamycin resistance. One such clone carrying the KanaR fragment at the ~laI site was denoted pFnb59.
i Fibronectin binding assa~
The fibronectin binding assay was a modification of the procedure !~
described~ by G. Froman et al, (JBC, 259, 14899-14905). Bacteria were inoculated on CFA plates for 42-48 hrs at 26C or 37C. Cells were resuspended in cold phosphate-buffered saline (pH 7.5) to ~n op~mal density of 109 cfu/ml. 100 ul of cells were added to an assay tube containing 1 ml of PBS + 0-1% Tween 80 + 100 /ul of l25I-fibronectin (5x105 cpm) and the .
mixture was end over incubated in room temperature for 1 hr. Tubes were centrifuged in Eppendorf centrifuge for five min. Supernatants were carefully 1, 8~BSTITUTE S~lEE-r wo 94/10330 pcr/uss3/1o547 1 ~
2 1 Q~ 8 l¦ rl {~ ~ ~

aspirated. The radioactivity in the pellet was measured in a liquid scintillation counter (LKB-Wallac).

Analvsis of protein ex~ression in minicells.
Plasmid constructs were transformed into the minicell-producing strain ~A10. Preparation and labelling of minicells with 35S-methionine were as 'r'' described by Thompson and Achtman ~Mol. Gen. Genet. 165, 295-304 (1978)).
The radioactive samples were separated on linear 15 % (wt/vol) SDS-polyacrylamide gels (Laemmli, UK, ~:~, 227, 880-885, (1970)). The gels were fixed, stained, destained, and exposed to X-ray film (DuPont) ~or 1-5 -days. Molecular weight standards were from Pharmacia Fine Chemicals, Uppsala, Sweden.
Precursor form of proteins encoded by different constructions were monitored after radiolabelling of minicells in the presence of 9% ethanol (Palva, J. Bact., 146, 325-330) and analyzed on SDS-polyacrylamide gels.

Electron microscopv.
Electron microscopy was perforrned using a JEOL 100B microscope with 100-mesh copper grids coated with thi~ films of 2% Formvar. Bacteria from CFA agar pla~es were resuspended in 10 rnM Tris-HC1,-pH 7.5 + 10 20 mM MgCl2, and placed on the grid. Grids were washed with buffer and negatively sta~ned for S sec. with 3.55 % ammonium molybdate, followed by washing with redistilled water. The present fibronec~n binding protein can be `, used ~or immunization, whereby the protein, preferably in combination with a fusion protein to create a large antigen to respond to, or the pili shaken off ~ -from ~e ~ coli expressing the curh pili when grown around about 30C, preferably at 26C, or the inactivated E. çQ~ comprising the curli pili ;1 consisting of the fibronectin binding 17 kD protein, is injected in dosages causing immunological reaction in the host mammal. Thus the fibronectin :' ~U--~T~ SY,~k ~

~Wo94/10330 2~ 17~ sg3/los47 ~

binding protein can be used in vaccination of ruminants against mastitis caused by E. coli infections. The fibronectin binding protein can further be used for ` -~`
immunization against urinary tract infections, norrnally caused by E. coli, or intestinal infections normally caused by E. coli, such as ulcerous colitis~ The 5 fibronectin binding protein of this invention has shown to form antibodies against E. coli related infections.
Further, the fibronectin binding protein can be used to block an infection in an open skin wound by wound treatment using the fibronectin binding protein in a suspension. Thus the fibronectin binding protein can be -used for the treatment of wounds, e.g., for blocking protein receptors, or for -immunization ~vaccination). In the latter case the host body produces specific `
antibodies which carl protect against invasion of bacterial strains comprising such a fibronectin binding protein. Hereby the antibodies block the adherence of the bacterial strains to damaged tissue.
lS Examples of colonizing of tissue damage are: -~
a) colonizing of wounds in skin and connective tissue, which wounds have been caused by a mechanical trauma, chemical damage, and/or thermical damage;
b) colonizing of wounds on mucous membranes such as in the mouth cavity, or in the mammary glands, urethra, or vagina;
c) colonizing on connective tissue proteins, which have been ex-posed by minimal tissue damage (micro lesion) in connection with epithelium ,,!
and endothelium (mastitis, heart valve infection, hip exchange surgery). g When using the present fnb protein or a synthesized amino acid ~ ;~
polypeptide for the purpose of immunization (vaccination) in mammals, including humans, the protein, or polypeptide, or curli pili, or whole ~ -inactivated bacteria, is dispersed in sterile, isotonic saline solution, optionally while adding a pharrnaceutieally acceptable dispersing agent. Different types of adjuvants can further be used in order to sustain the release in the tissue, 30 and thus expose the protein for a longer time to the immuno defense system of SUEiSTITUTE SHE~E~

WO 94/10330 PCr/US93~105~7 ~ ~ ~
2 `1 4 ~

a body. The injectable solutions will usually be given subcutaneously or intramuscularly .
A suitable dosage to obtain immuni~ation is 0.5 to 5 ug of fnb protein -per kg bodyweight and injection of immunization. In order to obtain durable 5 immuni~ation, vaccinations should be carried out at more than one consecutive occasions with an interval of 1 to 3 weeks, preferably at three occasions.
Whe!l using the present fnb protein for topical local administration the protein is dispersed in an isotonic saline solution to a concentration of 25 to 250 u per ml. The wounds are then treated with such an amount only to ;~
obtain a complete wetting of the wound surface. For an average wound thus , -only a couple of milliliters of solution are used in this way. After treatment using the protein solution the wounds are suitably washed with isotonic saline or another suitable wound treatment solu~ion.
Further the fibronecgin binding protein, or the synthesized polypeptide of the present invention can be used to diagnose bacterial infections caused by E. coli strains, whereby a fibronectin binding protein of the present invention is immobilized on a solid carrier, such as small latex or SepharoseR beads, whereupon sera containing antibodies are allowed to pass and react with the fibronectin binding protein thus immobilized. The agglutination is then measured by ~own methods.
Further the fibronectin binding protein or the polypeptide can be used in ELISA test (E. Engvall, "Enzyme Linked Immuno Sorbent A~ssay," Med.
Biol. C,5, 193, (1977)). Hereby wells in a polystyrene microtitre plates are coated with the fnb protein and incubated over night at 4C:. The plates are .
then thoroughly washed using PBS containing 0.05% Tween 20, and dried.
Serial dilution of the patient serum is made in PBS-Tween, are added to the i .
wells, and incubated at 30C for l.5 hrs. After rinsing antihuman-IgG
conjugated with an enzyme, or a horseradish peroxidase, or an alk~line phosphatase is added to the wells and incubated at 300C for 1.5 hrs., whereupon when tho IgG has been bound thereto, and after rinsing, an SU~.STITLDTE SHE~E'r , ... . .. ~ , . .. .. .. . . .

~; Wo 94/10330 Pcr/uss3/los~7 ~;
~ 21~ 3~17.9 ~

-19~
enzyrne substrate is added, a p-nitrophosphate in case of an alkaline phosphatase, or orthophenylene diamine substrate (OPD) in case a peroxidase ¦ ~
has been used, respectively. The plates comprising the wells are thus then ~ -rinsed using a citrate buffer containing O.OSS% OPD, and 0,005% H2O2, and S incubated at 30C for lO min. Enzyme reaction is stopped by adding a 4N `
solution of H2SO4 to each well. The color development is measured using a spectrophotometer .
Depending on the type of enzyme substrate used a fluorescence measurement can be used as well.
Another method to diagnose E. coli infections is by using the DNA
gene probe method based on the fnb protein sequence or the synthetic polypeptide sequence. Thereby the natural or synthetic DNA sequences are attached to a solid carrier, such as a polystyrene plate as mentioned above, by -~
e.g., adding a milk in the case of diagnosing a mastitis, to the surface. The DNA gene probe, optionally labelled enzyrmatically, or by a radioactive isotope is then added to the solid surface plate comprising the DNA sequence~ -~
whereby the DNA gene probe attaches to the sequence where appearing. The er~yme or the radioactive isotope can then readily be determined by known methods.
Above the tenn fibronectin binding protein includes any of the polypeptide sequences as well, which polypeptide se~uences ~orm the minimal fibronectin binding site of the comple~e protein.
,..
Fi~ure le~ends. ~ -Figure 1 Physical map of ~he different pFnb plasmid constructs. Fnb~ or - indicate ~:
fibronectin binding phenotype. Constructs~
pFnblO SphI,-KpnI ~ragment in pUCl8 ¦
`' 30 ClaI cut back of pFnblO
" 20 BgIII-SphI ~ragment in pUCl8 1, ~UB~TiTU, E S~IEEF

~?11 WO 94/10330 Pcr/vss3/lo~7 5;
` 2 ~ ~ 8 '1 1 ~ !: ~
,..j I .

" 56 SphI-HpaI " " " ~, " 59 APH Iigated to the ClaI2 site in pFnb56 " 46 ClaI-BglII fragment in pUCl8 Figure 2 S Restriction map and gene organization of fnb gene. The hatched boxes indicate the 17 kD.

Figure 3 , Nucleotide and predicted amino acid sequence of fnbA gene. Three stars indicate the stop codons.

".
, ~ 10 Electron microscopy pictures Figure 4 A012 26C
.1 Figure 5 HB101/pFnb56 26C
Figure 6 HB101/pFnb56 37C
.1 Figure 7 AA10/pFnb20 26C

l~,3 Electron microscopy was perforrned with a JOEL lOOB microscope with 100-mesh copper grids coated with thin films of 2% Forrnvar. Bacteria from CFA-agar plates were res~lspended in lOmM tris-HCl, pH 7,5-lOmM
MgCl2 and placed on the grid. ~ Grids were washed with buffer and negatively staine~ for S sec. with 3.55% ammonium molybate, followed by washing with 20 redistilled water.

SUBSTiTUT_ SHE'~

Claims (15)

CLAIMS:

1. A fibronectin binding protein appearing in the form of a po-lymer protein on the surface of E. coli in the form of curli pili, hereby defined as curlin.

2. A fibronectin binding protein according to claim 1, wherein the molecular weight of the protein is about 17kD.

3. Hybdd-DNA-molecule comprising a nucleotide sequence from E. coli coding for a protein or polypeptide having fibronectin binding ability.

4. Plasmid or phage comprising a nucleotide sequence from E.
coli coding for a protein or a polypeptide having fibronectin binding ability.

5. A plasmid pFnb2O as contained in the E. coli HB101 strain having the deposit number DSM 4585.

6. An E. coli strain expressing said fibronectin binding protein in the form of a curli pili, hereby defined as curlin.

7. A micro-organism transformed by recombinant DNA molecule of claims 3-5.

8. Hybrid-DNA-molecule according to claim 1, characterized in that it comprises one or more of the following nucleotide sequences

9. Plasmid or phage comprising one or more nucleotide sequences according to claim 8.

10. Micro-organism containing at least a plasmid or a phage ac-cording to claim 9.

11. A method for producing a fibronectin binding protein or polypeptide, wherein a) at least one hybrid-DNA-molecule according to claim 3 is introduced into a micro-organism, b) said microorganism is cultivated in a CFA growth medium at a temperature of 25-30°C for 35-50 hrs, and c) the protein thus expressed and formed as a pili is isolated.

12. A chemical synthesizing method for producing a fibronectin binding protein or polypeptide according to claims 1-3, whereby an amino acid residue is built up based on said nucleotide sequence encoding for said protein or polypeptide, starting from the C-terminal amino acid, alanine, which is stepwise reacted with the appropriate amino acid, whereby it is finally reacted with the amino acid at the N-terminal end, threonine, to form said fibronectin binding protein or polypeptide.

13. A fibronectin binding protein or polypeptide comprising at least the amino acid sequence

14. Pharmaceutical composition for the treatment of infections caused by E. coli, characterized in that it comprises a fibronectin binding protein according to claims 1-13 together with a pharmaceutically acceptable carrier.

15. Method for the treatment of infections caused by E. coli in mammals, whereby a pharmaceutically active amount of a fibronectin binding protein according to claims 1-13 is administered, optionally in combination with a pharmaceutically acceptable carrier.